Interferons (IFNS) are cytokine proteins that are produced by cells infected with viruses and that induce potent anti-viral activities via the immediate induction of gene expression following binding to cell surface receptors. Thus, interferons are not direct anti-viral agents, but induce one or several anti-viral mechanisms. In addition, interferons act on viruses other than the specific virus inducing the interferon.
The principal innate anti-viral mechanism for most cells involves the actions of Type I interferons (IFNα and IFNβ), leading to the induction of interferon responsive gene expression. Two families of transcriptional regulators, i.e., members of the “signal transducers and activators of transcription” (STAT) and “interferon regulatory factor” (IRF) families, work in conjunction to establish a cascade of gene regulation and signal transduction events that lead to transcriptional activation of interferon-stimulated genes (ISGs). Proteins encoded by such ISGs have potent anti-viral properties, which include disruption of the viral replicative life cycle, blockage of cell cycle progression, and apoptosis.
A trimeric complex, IFN Stimulated Gene Factor 3 (ISGF3), is formed following IFN binding to cell surface receptors and is comprised of (i) STAT1, (ii) STAT2, and (iii) p48/ISGF3γ/IRF9 (FIG. 3). The STAT family members are proteins of 100 kDa molecular weight containing highly conserved SH2 domains, SH3-like domains, and unique regions which serve as sites of interaction with other proteins involved in signal transduction. The STATs also contain general characteristics of transcription factors including a conserved DNA binding domain, a COOH-terminal transcriptional activation domain, and regions to contact other transcriptional regulators. The p48/ISGF3γ/IRF9 protein, herein referred to as p48, is not a STAT factor, but is a member of the IRF (Interferon Regulatory Factor) family. In addition, the p48 protein is an essential component of ISGF3 that contributes DNA binding specificity and provides specific protein binding sites for the recruitment of STAT1 and STAT2 proteins to the promoter, but is otherwise transcriptionally inert.
Genes that are transcriptionally activated by IFNs share a common promoter element called the IFN stimulated gene response element (ISRE); (AGTTTN3TTTCC, SEQ ID NO: 3). The trimeric protein complex, ISGF3, binds with high affinity to the ISRE following IFN treatment (Fu et al., 1990, Proc. Natl. Acad. Sci. USA, 87: 8555-8559; Fu et al., 1992, Proc. Natl. Acad. Sci. USA, 89: 7840-7843; Schindler et al., 1992, Proc. Natl. Acad. Sci. USA, 89: 7836-7839).
The general mechanism for activation of transcription by ISGF3 can be viewed as a two-phase process: As depicted in FIG. 4, the first phase (signaling phase) involves receptor-mediated signal transduction to generate a nuclear transcription complex; the second phase (transcription phase) involves the initiation of activation of target genes by the complex. The first phase has been well characterized. Binding of IFN to its receptor activates the tyrosine kinases JAK1 and TYK2 to phosphorylate the IFN receptor cytoplasmic domain, providing a docking site for STAT2. STAT2 phosphorylation provides a docking site for STAT1. Following STAT1 phosphorylation, the two STATs heterodimerize to form STAT1:2 heterodimer. Thereafter, the STAT1:2 heterodimer associates with p48 to form the trimeric ISGF3 complex. This trimeric complex represents an elaborate scheme to target the STAT2 C-terminus, which contains the essential transcriptional activation domain of ISGF3, to the appropriate promoters for participation in transcriptional activation.
Most transcription activating proteins contain a sequence-specific DNA binding domain linked to a transcriptional activation domain. Therefore, this modular nature of transcription factors is the basis of “two hybrid systems” for screening libraries for protein interaction partners. Indeed, it has been well documented that many protein regions can act as transcriptional activation domains (TADs) when fused to DNA binding domains, regardless of their original cellular function (see, e.g., Brent and Ptashne, 1985, Cell: 43: 729-36; Ma and Ptashne, 1987, Cell 51: 113-9; Ma and Ptashne, 1988, Cell 55: 443-6). While such TADs differ in the ability to mediate precise protein:protein contacts with transcriptional machinery, the TADs function similarly as activators of RNA polymerase.
IFNs are the principle innate anti-viral and anti-tumor cytokines and are also potent immuno-modulators that participate in the regulation of the functions of T-cells, B-cells, natural killer cells, and macrophages. Moreover, IFNs possess direct anti-proliferative activities and are cytostatic or cytotoxic for a number of different tumor and cancer cell types. Therefore, IFNs are involved in both anti-viral and anti-neoplastic (e.g., cancer and tumor) responses.
IFN genes were first cloned in 1979 and have been approved since 1991 for the treatment of hepatitis C infection. IFNs have been associated with the treatment of cancer and infectious diseases because of their roles in both the innate and adaptive immune systems. Specifically, IFNs have been employed for therapeutic use against hairy-cell leukemia, chronic hepatitis B, a major cause of liver cancer and cirrhosis, as well as for treatment of genital warts and some rarer cancers of the blood and bone marrow. Nasal sprays containing alpha interferon also provide some protection against colds caused by rhinoviruses.
However, as the IFN system represents an early and crucial step in anti-viral immunity, it is not surprising to find that many viruses have evolved strategies to block the actions of IFN. The ability of a virus to antagonize the IFN pathway can have dramatic consequences for the success of infection. The virulence of a specific virus strain can be determined largely by susceptibility to the anti-viral effects of IFN. Furthermore, the mutations which enhance IFN resistance can lead to highly infectious and persistent infections. The ability of a wide variety of viruses to fight the IFN system is illustrated by the diverse strategies used to overcome the effects of IFN (Bergmann et al., 2000, Journal of Virology; 74(13): 6203-6206 and Kitajewski et al., 1986, Cell; 45(2): 195-200). The viral proteins block a variety of steps in the IFN signaling system, in many cases at an early point upstream of gene activation.
Accordingly, there exists a need to activate interferon stimulated gene expression directly by bypassing the normal interferon induced pathway for the transcription of ISGs. Consequently, the virus families which have evolved strategies to block the actions of IFN will not have the ability to hinder the IFN pathway, since the interferon stimulated genes will be activated directly even in the absence of IFN binding to its receptor. In addition, cancer cells and tumor cells which have developed mechanisms to evade IFN action may also benefit by the direct activation of interferon stimulated genes. The present invention satisfies such a need by providing fusion protein transcription regulators which provide effective gene therapy strategies for virus infections as a result of activating transcription of interferon stimulated genes directly, thus bypassing the need of IFN for ISG-gene expression.